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Research Article

Synthesis and biological evaluation of novel propargylquinobenzothiazines and their derivatives as potential antiproliferative, anti-inflammatory, and anticancer agents

Małgorzata JeleńDepartment of Organic Chemistry, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Sosnowiec, Katowice, Poland and
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Krystian PlutaDepartment of Organic Chemistry, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Sosnowiec, Katowice, Poland and Correspondence[email protected]
,
Michał ZimeckiDepartment of Experimental Therapy, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
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Beata Morak-MłodawskaDepartment of Organic Chemistry, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Sosnowiec, Katowice, Poland and
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Jolanta ArtymDepartment of Experimental Therapy, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
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Maja KociębaDepartment of Experimental Therapy, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
&
Iwona KochanowskaDepartment of Experimental Therapy, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
 show all
Pages 83-88 | Received 22 Apr 2016, Accepted 15 Jun 2016, Published online: 12 Jul 2016

Abstract

Azaphenothiazines containing the quinoline ring, 8–10-substituted 6H-quinobenzothiazines and 6H-diquinothiazine were transformed into new 6-propargyl and 6-dialkylaminobutynyl derivatives containing the triple bond. Most of them displayed strong antiproliferative actions against human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin A (PHA), strongly suppressed lipopolysaccharide (LPS)-induced TNF-α production by whole blood human cell cultures, and exhibited low cytotoxicity. Three propargylquinobenzothiazines with the bromine, trifluoromethyl, and methylthio groups at position 9 and propargyldiquinothiazine exhibited comparable actions to cisplatin against the L-1210 and SW-948 tumor lines. 6-Propargyl-9-trifluoromethylquinobenzothiazine was shown to block caspase 3 expression and inhibit expression of caspase 8 and 9 in Jurkat cells indicating its possible mechanism of action. These derivatives could be promising, potential therapeutics for treatment of neoplastic diseases and autoimmune disorders.

Introduction

Classical tricyclic phenothiazines have been known for years as a source of effective neuroleptic, antihistaminic, antitussive, and antiemetic drugsCitation1. Their N-substituted dibenzo-1,4-thiazine system was found to be a valuable molecular template for the development of new derivatives exhibiting a variety of new types of activities. Numerous reports describe new promising activities of classical and modified phenothiazines: such as anticancer, multidrug reversal, antibacterial, antifungal, anti-inflammatory, analgesic, antiviral, antimalarial, immunosuppressive, antifilarial, and trypanocidal. The compounds are also regarded to have potential benefits in the treatment of Alzheimer’s, Creutzfeldt–Jakob’s, and AIDS-associated diseases. These promising and valuable activities have been reviewed in numerous papers and chaptersCitation2–15.

New phenothiazine derivatives were obtained by several approaches, such as introduction of new substituents to the nitrogen atom or to the benzene ring, oxidation of the sulfur atom to the sulfoxide and sulfone function, and by changing one or two benzene rings with a homoaromatic (e.g. naphthalene) and heteroaromatic rings (pyridine, pyridazine, pyrimidine, pyrazine, quinoline, and quinoxaline). The introduction of the azine ring led to the formation of a large group of phenothiazine derivatives, azaphenothiazines of the heteroarenobenzothiazine, and diheteroarenothiazine structures. About 40 types of azaphenothiazines are known as monoaza-, diaza-, triaza-, and tetraazaphenothiazines and their monobenzo- and dibenzo derivativesCitation16. Some of these azaphenothiazines being pyridobenzothiazinesCitation17–21, pyrimidobenzothiazinesCitation22,Citation23, pyrazinobenzothiazinesCitation24,Citation25, dipyridothiazinesCitation26–30, quinobenzothiazinesCitation31–36, quinobenzothiazinium saltsCitation37–39, diquinothiazinesCitation26,Citation27, and quinonaphthothiazinesCitation36,Citation40 exhibited promising anticancer, antibacterial, antiviral, immunosuppressive, antioxidative anti-inflammatory, and enzyme inhibiting activities.

Encouraged by obtaining compounds of promising anti-proliferative, anticancer, and anti-inflammatory properties of 9-fluoro-6-propargylquinobenzothiazine 1Citation35, we synthesized new propargylazaphenothiazines: 6-propargylquinobenzothiazines with the substituents in positions 8, 9, and 10, and 6-propargyl diquinobenzothiazine. As most of the biologically active phenothiazine derivatives possess the tertiary amine group, we transformed a selected propargyl compound into the N, N-dialkylaminomethyl derivatives. Then, we evaluated their toxicities with regard to human blood lymphocytes, suppressive actions on mitogen-induced lymphocyte proliferation, lipopolysaccharide-induced TNF-α production in human whole blood culture, and in vitro tumor cell growth. Lastly, we attempted to established effects of one representative compound on expression of caspases and DNA fragmentation in Jurkat cells.

Methods

Chemistry

Melting points were determined in open capillary tubes in a Boetius melting point apparatus and were uncorrected. The NMR spectra were recorded on Bruker Fourier 300 and Bruker DRX spectrometers (Bruker Optics, Billerica, MA) (1H at 300 and 500 MHz) in CDCl3. Electron impact (EI MS) and chemical ionization (CI MS) mass spectra were run on a Finnigan MAT 95 spectrometer (Thermo Finnigan LLC, San Jose, CA) at 70 eV. Elemental analysis was run on a Perkin-Elmer 2400 analyzer (Perkin-Elmer Inc., San Deigo, CA).

8–10-Substituted 6H-quinobenzothiazines (4a–h) and 6H-diquinothiazine (9) were prepared as described previously using diquinodithiins (2 and 7) and 2,2′-dichloro-3,3′-diquinolinyl sulfide (8) and disulfide (3) followed the procedure described in Refs. [Citation41–43].

Synthesis of propargyl derivatives 5a–h and 10

To a solution of 6H-quinobenzothiazine (4a–h) or 6H-diquinothiazine (9) (1 mmol) in dry DMF (5 mL) potassium tert-butoxide (0.16 g, 1.44 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. Then 80% solution of propargyl bromide in toluene (0.3 mL, 2.7 mmol) was added dropwise and the stirring was continued for 24 h. The reaction mixture was poured into water (25 mL). The resulting solid was filtered off, washed with water and purified by column chromatography (silica gel, CH2Cl2) to give the following (Scheme 1):

  1. 6-Propargylquinobenzothiazine 5a (0.21 g, 73.0%), mp 141–142 °C (EtOH). 1H NMR (CDCl3) δ: 2.31 (t, 1H, CH), 5.05 (d, 2H, CH2), 7,00 (t, 1H, H9), 7.14 (d, 1H, H7), 7.25 (m, 2H, H8, H10), 7.31 (d, 1H, H2), 7.54 (d, 1H, H3), 7.55 (d, 1H, H1), 7.72 (s, 1H, H12), 7.95 (d, 1H, H4). EI MS m/z: 289 (M + 1, 100), 250 (M − C3H4, 20). Anal. Calcd for C18H12N2S: C, 74.97; H, 4.19; N, 9.71. Found C, 74.82; H, 4.21; N, 9.55.

  2. 8-Chloro-6-propargylquinobenzothiazine 5b (0.23 g, 72.1%), mp 170–171 °C (EtOH). 1H NMR (CDCl3) δ: 2.35 (t, 1H, CH), 4.99 (d, 2H, CH2), 6,98 (m, 1H, H7), 7.05 (m, 1H, H9), 7.25 (d, 1H, H10), 7.35 (d, 1H, H2), 7.56 (m, 2H, H1, H3), 7.71 (s, 1H, H12), 7.89 (d, 1H, H4). CI MS m/z: 322 (M+, 25), 283 (M − C3H3, 15). Anal. Calcd for C18H11ClN2S: C, 66.97; H, 3.43; N, 8.68. Found C, 66.79; H, 3.40; N, 8.53.

  3. 9-Chloro-6-propargylquinobenzothiazine 5c (0.24 g, 75.2%), mp 169–170 °C (EtOH). 1H NMR (CDCl3) δ: 2.32 (t, 1H, CH), 5.05 (d, 2H, CH2), 7,13 (d, 1H, H7), 7.16 (d, 1H, H10), 7.19 (m, 1H, H8), 7.34 (t, 1H, H2), 7.57 (m, 2H, H1, H3), 7.74 (s, 1H, H12), 7.80 (d, 1H, H4). EI MS m/z: 322 (M+, 100), 283 (M − C3H3, 50). Anal. calcd for C18H11ClN2S: C, 66.97; H, 3.43; N, 8.68. Found C, 66.81; H, 3.42; N, 8.56.

  4. 9-Bromo-6-propargylquinobenzothiazine 5d (0.26 g, 71.0%), mp 192–193 °C (EtOH). 1H NMR (CDCl3) δ: 2.31 (t, 1H, CH), 4.96 (d, 2H, CH2), 7,11 (d, 1H, H7), 7.27 (d, 1H, H10), 7.33 (m, 2H, H2, H8), 7.54 (m, 2H, H1, H3), 7.70 (s, 1H, H12), 7.84 (d, 1H, H4). CI MS m/z: 368 (M + 1, 80), 229 (M − C3H3, 40). Anal. Calcd for C18H11BrN2S: C, 58.87; H, 3.02; N, 7.63. Found C, 58.69; H, 3.00; N, 7.47.

  5. 9-Trifluoromethyl-6-propargylquinobenzothiazine 5e (0.25 g, 69.4%), mp 149–150 °C (EtOH). 1H NMR (CDCl3) δ: 2.33 (t, 1H, CH), 4.99 (d, 2H, CH2), 7.30 (d, 1H, H7), 7.34 (m, 2H, H8, H10), 7.46 (d, 1H, H2), 7.55 (m, 2H, H1, H3), 7.70 (s, 1H, H12), 7.83 (d, 1H, H4). EI MS m/z: 356 (M+, 100), 317 (M − C3H3, 35). Anal. calcd for C19H11F3N2S: C, 64.04; H, 3.11; N, 7.86. Found C, 63.84; H, 3.08; N, 7.69.

  6. 9-Methoxy-6-propargylquinobenzothiazine 5f (0.24 g, 75.5%), mp 148–149 °C (EtOH). 1H NMR (CDCl3) δ: 2.30 (t, 1H, CH), 3.74 (s, 3H, CH3), 4.96 (d, 2H, CH2), 6.73 (d, 1H, H7), 6.78 (d, 1H, H8), 7.19 (d, 1H, H10), 7.27 (t, 1H, H2), 7.52 (t, 1H, H3), 7.55 (d, 1H, H1), 7.68 (s, 1H, H12), 7.82 (d, 1H, H4). CI MS m/z: 318 (M+, 95), 303 (M − CH3, 65), 279 (M − C3H3, 100). Anal. calcd for C19H14N2OS: C, 71.67; H, 4.43; N, 8.80. Found C, 71.49; H, 4.40; N, 8.59.

  7. 9-Methylthio-6-propargylquinobenzothiazine 5 g (0.25 g, 74.9%), mp 154–155 °C (EtOH). 1H NMR (CDCl3) δ: 2.31 (t, 1H, CH), 2.48 (s, 3H, CH3), 5.02 (d, 2H, CH2), 7.07 (d, 1H, H7), 7.16 (m, 2H, H8, H10), 7.32 (t, 1H, H2), 7.55 (m, 2H, H1, H3), 7.71 (s, 1H, H12), 7.90 (d, 1H, H4). EI MS m/z: 334 (M+, 100), 295 (M − C3H3, 75). Anal. calcd for C19H14N2S2: C, 68.23; H, 4.22; N, 8.38. Found C, 68.02; H, 4.25; N, 8.19.

  8. 10-Chloro-6-propargylquinobenzothiazine 5 h (0.24 g, 75.0%), mp 162–163 °C (EtOH). 1H NMR (CDCl3) δ: 2.32 (t, 1H, CH), 5.01 (d, 2H, CH2), 7,06 (d, 1H, H7), 7.15 (m, 2H, H8, H9), 7.36 (m, 1H, H2), 7.55 (m, 2H, H1, H3), 7.57 (s, 1H, H12), 7.67 (d, 1H, H4). EI MS m/z: 322 (M+, 100), 283 (M − C3H3, 40). Anal. calcd for C18H11ClN2S: C, 66.97; H, 3.43; N, 8.68. Found C, 66.78; H, 3.44; N, 8.45.

  9. 6-Propargyldiquinothiazine 10 (0.27 g, 79.6%), mp 212–213 °C (EtOH). 1H NMR (CDCl3) δ: 2.15 (t, 1H, CH), 5.59 (d, 2H, CH2), 7,36 (m, 2H, H2, H10), 7.58 (m, 4H, H1, H3, H9, H11), 7.76 (s, 2H, H12, H14), 7.93 (d, 2H, H4, H8). CI MS m/z: 339 (M+, 60), 338 (M − 1, 65), 300 (M − C3H3, 10). Anal. calcd for C21H13N3S: C, 74.31; H, 3.86; N, 12.38. Found C, 74.23; H, 3.83; N, 12.20.

Scheme 1. Synthesis of quinobenzothiazines 4–6 and diquinothiazines 9 and 10.

Scheme 1. Synthesis of quinobenzothiazines 4–6 and diquinothiazines 9 and 10.

Synthesis of dialkylaminobutynyl derivatives 6a–c

A mixture of 9-methylthio-6-propargylquinobenzothiazine (5 g) (0.17 g, 0.5 mmol), paraformaldehyde (0.03 g, 0.5 mmol), amine (diethylamine, pyrrolidine or piperidine, 0.7 mmol (0.05 g, 0.03 mL, 0.07 mmol), and copper(I) chloride (catalytic amount) in peroxide-free, dry dioxane (10 mL) was heated with continuous stirring at 50–55 °C (when diethylamine used) or 70–80 °C (when pyrrolidine or piperidine used) for 3 h. After cooling, 20 ml water was added and the mixture was extracted with chloroform, dried with Na2SO4, and evaporated in vacuo. The dry residue was dissolved in CHCl3 and purified by column chromatography (aluminum oxide, CHCl3) to give the following:

  1. 6-(4-dietylaminobut-2-ynyl)-9-methylthioquinobenzothiazine (6a) (0.33 g, 78.8%), an oil. 1H NMR (CDCl3) δ: 1.02 (t, 6H, 2CH3), 2.47 (s, 3H, CH3), 2.53 (q, 4H, 2CH2), 3.43 (t, 2H, CH2), 4.94 (s, 2H, CH2), 5.02 (s, 2H, CH2), 7.07 (d, 1H, H7), 7.15 (m, 2H, H8, H10), 7.28 (t, 1H, H2), 7.51 (m, 2H, H1, H3), 7.67 (s, 1H, H12), 7.77 (d, 1H, H4). CI MS m/z: 419 (M + 1, 75), 347 (M-NC4H10, 100). Anal. Calcd for Calcd for C24H25N3S2: C, 68.70; H, 6.01; N, 10.01. Found C, 68.64; H, 6.13; N, 10.22.

  2. 6-(4-Pyrrolidin-1-yl-but-2-ynyl)-9-methylthioquinobenzothiazine (6b) (0.33 g, 79.1%), mp 114–115 °C. 1H NMR (CDCl3) δ: 1.74 (m, 4H, 2CH2), 2.47 (s, 3H, CH3), 2.60 (m, 4H, 2CH2), 3.43 (s, 2H, CH2), 4.95 (s, 2H, CH2), 7.05 (d, 1H, H7), 7.15 (m, 2H, H8, H10), 7.28 (t, 1H, H2), 7.51 (m, 2H, H1, H3), 7.65 (s, 1H, H12), 7.76 (d, 1H, H4). CI MS m/z: 417 (M + 1, 80), 347 (M-NC4H8, 100). Anal. Calcd for C24H23N3S2: C, 69.03; H, 5.55; N, 10.06. Found C, 69.26; H, 5.63; N, 10.03.

  3. 6-(4-Piperidin-1-yl-but-2-ynyl)-9-methylthioquinobenzothiazine 6c (0.36 g, 83,7%), mp 132–133 °C. 1H NMR (CDCl3) δ: 1.35 (m, 2H, CH2), 1.58 (m, 4H, 2CH2), 2.47 (m, 7H, 2CH2, CH3), 3.38 (m, 2H, CH2), 4.96 (s, 2H, CH2), 7.07 (d, 1H, H7), 7.16 (m, 2H, H8, H10), 7.29 (t, 1H, H2), 7.52 (m, 2H, H1, H3), 7.67 (s, 1H, H12), 7.77 (d, 1H, H4). CI MS m/z: 431 (M + 1, 80), 347 (M − NC5H10, 100). Anal. Calcd for C25H25N3S2: C, 69.57; H, 5.84; N, 9.74. Found C, 69.38; H, 5.73; N, 9.55.

Biological assays

Reagents

Fetal calf serum (FCS), RPMI-1640 and Hanks’ medium were purchased from CytoGen GmbH (Sinn, Germany). Lipopolysaccharide from Escherichia coli 0111:B4 (LPS), phytohemagglutinin (PHA), dimethyl sulfoxide (DMSO), and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and all other reagents were from Sigma-Aldrich (St. Louis, MO).

Preparation of the compounds for biological assays

The compounds were dissolved in DMSO (10 mg/mL) and subsequently diluted in RPMI-1640 cell culture medium.

Isolation of the peripheral blood mononuclear cells (PBMC)

Venous blood from a single donor was withdrawn into heparinized syringes and diluted twice with PBS. PBMC were isolated by centrifugation on Ficoll–Uropoline gradient (density 1.077 g/mL) at 800 × g for 20 min at 4 °C. The interphase cells were then washed three times with Hanks’ medium and re-suspended in a culture medium, referred to below as the culture medium, consisting of RPMI-1640, supplemented with 10% fetal calf serum, l-glutamine, sodium pyruvate, 2-mercaptoethanol and antibiotics, at density of 2 × 106 cells/mL.

Cytotoxicity of the compounds against human PBMC

PBMC were washed three times with Hanks’ medium and re-suspended in the culture medium at density of 2 × 106 cells/mL. 2 × 105 cells/100 μL/well were incubated with the tested compounds at doses 10 and 1 μg/mL for 24 h in a cell culture incubator. Then, the cell viability was evaluated using MTT colorimetric methodCitation44. The results, originally calculated as optical density (OD) values, were presented as percent inhibition in comparison to appropriate DMSO control.

PHA-induced proliferation of human PBMC

PBMC were distributed into 96-well flat-bottom plates in 100 μL aliquots (2 × 105 cells/well). PHA was added at a concentration of 5 μg/mL. The compounds were tested at doses of 10 and 1 μg/mL. DMSO at appropriate dilutions served as control. After 4-d incubation in a cell culture incubator, the proliferative response of the cells was determined by the colorimetric MTT methodCitation44. The results, originally calculated as OD values, were presented as percent inhibition in comparison with appropriate DMSO control.

Lipolysaccharide-induced TNF-α production in whole blood cell culture

Human whole blood was diluted 5 × with RPMI 1640 medium and distributed to 24-well culture plates in 1 mL aliquots. The cultures were stimulated with LPS (1 μg/mL) and the studied compounds were added at a concentrations of 5, 1, and 0.1 μg/mL. The control cultures contained DMSO in appropriate concentration. After an overnight incubation the supernatants were harvested and frozen at −80 °C until cytokine determination. TNF-α levels were determined in the supernatants by using ELISA kit from eBioscience (San Diego, CA), in the presence of TNF-α standard, and originally expressed in pg/mL. The inhibition of TNF-α production (in percentage) was calculated from comparison with DMSO controls and was presented in the table.

Growth inhibition of tumor cell lines

L-1210 lymphoma and SW-948 colon tumor cell lines derived from the collection of cell lines of the Institute of Immunology and Experimental Therapy, Wrocław, Poland. The lines were re-suspended in the culture medium and distributed into 96-well flat-bottom culture plates (Nunc, Rochester, NY). L-1210 cells were incubated at 1.5 × 104 cells/100 μL/well while SW948 at 2.5 × 104 cells/100 μL/well. The preparations were added to the cultures at the concentration range 50–3.125 μg/mL. After 72 h incubation in a cell culture incubator the cell viability was determined using MTT colorimetric method and originally expressed as OD valuesCitation44. The results are presented as GI50 as calculated from comparisons with DMSO controls.

Colorimetric MTT assay for cell growth and kill

The assay was performed according to Hansen et al.Citation44. Briefly, 25 μL of MTT (5 mg/mL) stock solution was added per well at the end of cell incubation period and the plates were incubated for additional 3 h in a cell culture incubator. Then, 100 μL of the extraction buffer (20% SDS with 50% DMF, pH 4.7) was added. After an overnight incubation, the OD was measured at 550 nm with the reference wavelength of 630 nm in a Dynatech 5000 spectrophotometer (Dynex Technology, Chantilly, VA).

Cultures of Jurkat cells and total RNA isolation

Jurkat cells (105/mL) in the culture medium were cultured overnight with MZO-2 (25 μg/mL) Total RNA isolation was carried with TRIzol Reagent (Ambion, Austin, TX) according to recommendations of the manufacturer. The cell pellet (2 × 106 cells) was suspended in 1 mL of TRIzol reagent, shaken, incubated for 10 min at room temperature (RT), supplemented with 0.2 mL of chloroform, shaken vigorously for 15 s, incubated for 3 min at RT, and centrifuged at 12 000 × g for 15 min at 4 °C. The water phase was collected, transferred to a new tube, supplemented with 0.5 ml of isopropanol, incubated at RT for 10 min, and centrifuged at 12 000 × g for 10 min at 4 °C. The RNA pellet was washed with 1 mL of 75% ethanol, dried in air and dissolved in 20–30 μL of sterile diethylpyrocarbonate-treated Mili-Q water. RNA samples were stored at −20 °C.

Reverse transcription

Single stranded complementary DNA (cDNA) was synthesized with oligo (dT)12–18 primers from 5 μg of total RNA using Novazym VerteKit (Thermo Fisher Scientific Inc., Waltham, MA), accordingly to the instruction of the manufacturer.

Quantitation of gene expression by real-time PCR

Expression of the genes, i.e. β-actin, caspase 3, caspase 8, and caspase 9, was measured using APA SYBR FAST qPCR Kit. The sequences of primers are enclosed in the Supplementary material. The reaction was performed in Applied Biosystems ViiA7 thermocycler (Waltham, MA) starting with 5 min of preincubation at 95 °C followed by 35 amplification cycles as follows: 95 °C for 30 s and simultaneous annealing-extension-data acquisition for 45 s and 60 °C. Beta-actin was used as a housekeeping gene for arbitrary unit calculation for every tested gene.

Statistics

The results are presented as mean values ± standard error (SE). Brown–Forsyth’s test was used to determine the homogeneity of variance between groups. When the variance was homogenous, analysis of variance (one-way ANOVA) was applied, followed by post hoc comparisons with Tukey’s test to estimate the significance of the difference between groups. Nonparametric data were evaluated with Kruskal–Wallis’ analysis of variance. Significance was determined at p < 0.05. Statistical analysis was performed using STATISTICA 6.1 for Windows (STAT Inc., Cary, NC).

Results and discussion

Chemistry

The tetracyclic substituted 6H-quinobenzothiazine (4) was obtained in the original reactions of diquinodithiin (2, 5,12-diaza-6,13-dithiapentacene) and 2,2′-dichloro-3,3′-diquinolinyl disulfide (3) with aniline and its substituted derivatives (m-, and p-Cl, p-Br, p-CF3, p-OCH3, p-SCH3) as described by Jeleń and PlutaCitation41. The pentacyclic 6H-diquinothiazine (9) was synthesized in the reactions of diquinodithiin (7, 5,7-diaza-6,13-dithiapentacene) and 2,2′-dichloro-3,3′-diquinolinyl sulfide (8) with acetamide, followed the procedure described by Nowak et al.Citation42 and Jeleń and PlutaCitation43.

8–10-Substituted 6H-quinobenzothiazines (4a–h) and 6H-diquinothiazine (9) were N-propargylated with propargyl bromide in anhydrous DMF in the presence of potassium t-butoxide to give the propargyl derivatives (5a–h, 10) with good yield (69.4–79.6%). The selected propargyl compound (5g) was undergone the Mannich reaction with formaldehyde and cyclic and acyclic secondary amines to give the dialkylaminobutynyl derivatives (6a–c) with 78.8–83.7% yield.

The analysis of the proton signals in the quinobenzothiazine and diquinothiazine system in 1H NMR spectra of the products (5) and (10) pointed at the N-alkylation at the thazine nitrogen atom.

Biological activities

The compounds were tested for their cytotoxic effects against unstimulated human PBMC, inhibitory actions on PHA-stimulated proliferation of PBMC and LPS-induced TNF-α production in human whole blood cultures (). The cytotoxic properties were differential depending on the compound’s structures. All compounds exhibited low or very low toxicity at 10 μg/mL. The least cytotoxic were propargylquinobenzothiazine with the trifluoromethyl group (5e) and piperazinylbutynyl-9-methylquinobenzothiazine (6c), not exceeding 10%. Only propargyl derivatives (5a and 5c), without additional substituent and with the chlorine atom, exhibited toxicity over 30%. The isomeric chloro compounds (5b, 5c, and 5h) exhibited slightly different cytotoxicity depending on the place of that substituent. Nearly all compounds were completely devoid of toxicity at 1 μg/mL. Most of the compounds exhibited strong antiproliferative action in PHA-stimulated PBMC exceeding over 69% inhibition at 10 μg/mL (except 5e), and moderate suppressive actions within the range of 20.2–56.7%, still at concentration of 1 μg/mL.

Table 1. Effects of the compounds on PBMC survival, PHA-induced blood lymphocyte proliferation and TNF-α production by whole blood cultures. NT, not toxic.

All compounds displayed also strong or very strong inhibitory actions on LPS-induced TNF-α production in whole blood cultures at 5 and 1 μg/mL with a still marked inhibition at 0.1 μg/mL ().

For the evaluation of their anticancer properties, we selected compounds (5d, 5e, 5g, and 10) showing low cytotoxicity, combined with strong inhibitory actions with regard to PHA-induced PBMC proliferation, and tested them for growth inhibition of L-1210 leukemia and SW-948 tumor cell lines at 50–3.125 μg/mL concentration range. CisplatinCitation45, a commonly used antitumor agent, served as a reference drug. depicts the IC50 values of the investigated compounds. It appeared that compound 5d was more active than cisplatin against the SW-948 cell line. Other compounds showed comparable anticancer action against SW-948 line as the reference drug-cisplatin. Compounds 5d, 5f, and 10 exhibited strong anticancer activity against the L-1210 cell line but a few times weaker than the reference drug. A comparison of the antiproliferative, cytotoxic, and TNF-α inhibiting activity of propargylazaphenothiazines, diquinobenzothiazine 10 with qinobenzothiazine 5a, suggests that the introduction of the quinoline ring instead of the benzene ring to the azaphenothiazine system decreases the cytotoxic action.

Table 2. Inhibitory effects of the compounds on growth of SW-984 and L-1210 cell lines. The results represent mean IC50 values from three independent experiments.

Molecular studies on the mechanism of action

Very potent suppressive actions of the compounds on PHA-induced PBMC prolifeartion and LPS-induced TNF-α production prompted us determine expression of caspases, involved in activation of T cellsCitation46 in Jurkat and L-1210 cell lines. The results () showed that compound 5e, at a dose of 10 μg/ml, completely inhibited the expression of caspase 3 and lowered the expressions of caspases 8 and 9 in 24 h culture of Jurkat cells. β-actin RNA served as a reference housekeeping gene. The expression of all caspases was also inhibited in L-1210 cell line. On the contrary, the compound did not cause DNA fragmentation in Jurkat cells (data include in the Supplementary material) indicating lack of apoptosis induction.

Table 3. Effect of 5e compound on expresion of caspases in Jurkat and L-1210 cell lines.

Conclusion

6H-quinobenzothiazines (4) and 6H-diquinothiazine (10) were transformed into new derivatives (5, 6, and 10) with the triple bond: 6-propargylquinobenzothiazines with the additional substituents at position 8–10 (5a–h), 6-dialkylaminobutynyl-9-methylthioquinobenzothiazines (6a–c), and 6-propargyldiquinothiazine (10). Most of those compounds showed very strong antiproliferative and TNF-a inhibiting activity. Four selected compounds were subsequently tested for their antitumor action. The most active was compound 5d with the propargyl and bromine substituents. It showed very strong antiproliferative action and better suppressive actions than cisplatin on growth of SW-948 tumor cell line. This compound and two other (5g, 10) showed also strong action against L-1210 cell line but slightly weaker than the reference drug.

The demonstration of inhibition of caspase expression in Jurkat cell by 5e peptide provides explation for its suppressive effect in PHA-induced proliferation of PBMC since caspases are essential for IL-2 release upon T cell activationCitation46. In addition, T-cell receptor-induced NF-κB activation and subsequent IL-2 production is also dependant on caspase cascadeCitation47. The inhibition of caspase expression, most probably associated with inhibition of NF-κB activity, could also explain strong inhibition of TNF-α production by these compounds. Such a mechanism of action may be common for other phenothiazines since mepasine, a phenothiazine derivative, suppressed symptoms of multiple sclerosis in mice by inhibition of MALT 1, a paracaspase crucial for lymphocyte activation via NF-κBCitation48. Interestingly, phenothiazines can also kill cancer cells by means of other mechanisms, for example by lysosome-dependent autophagyCitation49. In addition, the deep inhibition of inducible TNF-α, a major mediator of inflammationCitation50, strongly suggests that the investigated compounds may diminish inflammatory processes in vivo.

In summary, the compounds may be of interest as potential antitumor, antiproliferative, and anti-inflammatory therapeutics. Their value is further enhanced by low cytotoxicity. Further studies will be aimed at the mechanism of antitumor action and evaluation of therapeutic utility of these compounds in several mouse models in vivo such as allergy, chemically induced colitis and allogeneic skin transplantation.

Declaration of interest

The work is supported by The Medical University of Silesia (Grant KNW-1–004/K/4/0).

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Synthesis and biological evaluation of novel propargylquinobenzothiazines and their derivatives as potential antiproliferative, anti-inflammatory, and anticancer agents
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Synthesis and biological evaluation of novel propargylquinobenzothiazines and their derivatives as potential antiproliferative, anti-inflammatory, and anticancer agents
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